Discovery of small molecules against porcine reproductive and respiratory syndrome virus replication by targeting NendoU activity

Abstract

Porcine reproductive and respiratory syndrome (PRRS) remains a major threat to animal health and causes substantial economic losses worldwide. The nonstructural protein 11 (NSP11) of the causative agent, PRRS virus (PRRSV), contains a highly conserved nidoviral uridylate-specific endoribonuclease (NendoU) domain essential for viral replication and immune evasion. Targeting NSP11 offers a novel approach to antiviral intervention. Through in silico virtual screening followed by a fluorescence resonance energy transfer assay , we identified A8-A2 as a promising candidate that effectively inhibits NendoU activity. Molecular docking and mutational analysis revealed that A8-A2 and its analogs target the key catalytic residues His144 and Thr217 of NSP11, located within the NendoU enzyme activity loop and pocket region, respectively. A8-A2 demonstrated dose-dependent inhibition of PRRSV replication in porcine alveolar macrophages. Notably, the NendoU is conserved across PRRSV strains and other Nidoviruses, and A8-A2 exhibited antiviral activity against both type I and type II PRRSV strains, as well as the infectious bronchitis virus, a coronavirus in the order Nidovirales. Further investigations revealed that A8-A2 impedes viral replication early in infection and reverses NSP11-mediated suppression of Poly(I:C)-induced interferon production. However, this effect occurs independently of mRNA splicing inhibition. These findings indicate that A8-A2 could act as an effective antiviral agent against infections caused by diverse PRRSV strains and may serve as a broad-spectrum agent for other Nidoviruses.

Importance: Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the pig industry, and vaccination is the principal method to prevent this viral infection currently. However, vaccination often fails to provide protection against heterologous strains, highlighting the need for alternative strategies for broad protection. The nidoviral uridylate-specific endoribonuclease (NendoU) domain plays a crucial role in viral replication and evasion of host immune responses. In this study, we identified a group of new compounds with similar chemical structures that could interfere with NendoU enzyme activity. Among these compounds, A8-A2 significantly inhibited PRRSV replication in host cells with minimal cytotoxicity. Our findings provide a new direction for developing potent antiviral compounds that can offer broad protection against different PRRSV strains, thereby mitigating their impact on pig health and benefiting the husbandry industry.

Keywords: NendoU; PRRSV; antiviral agents; nidovirus.

Fig 1

Screening for inhibitors of PRRSV NSP11 NendoU activity. NendoU activity analysis of VR-NSP11 (A) and LY-NSP11 (B). Different concentrations of VR-NSP11 or LY-NSP11 protein were incubated with R23.1 oligos to measure the endoribonuclease activity every 20 min. DsRed: negative protein control. DsRed were incubated with R23.1 oligo. Values represent mean ± SD, n = 3. NendoU activity inhibition analysis for VR-NSP11 (C) and LY-NSP11 (D). 1.2 µM VR-NSP11 and 0.52 µM LY-NSP11 were incubated with R23.1 oligo and each of the 33 compounds at 15 µM level. NC: negative vehicle control with NSP11 and R23.1 oligo mixed with DMSO; DsRed: negative protein control with DsRed and R23.1 oligo mixed with DMSO. Values represent mean ± SD, n = 3. (E) Chemical structures of compounds A8, B4, and C5. (F) Dosage-dependent inhibitory activity of compound C5 to NSP11 at 2 h. Values represent mean ± SD, n = 3. (G) Cytotoxicity analysis of A8, B4, and C5. Compounds were incubated with PAMs for 48 h, with DMSO as the vehicle control. The red dashed line indicates 95% relative viability. The top x-axis illustrates DMSO concentration, expressed as the ratio of DMSO volume to medium volume (vol/vol). The bottom x-axis denotes compound concentrations (µM). Values represent mean ± SD, n = 3.

Fig 2

Assessment of the cytotoxicity of NSP11 inhibitor analogs. NendoU activity inhibition analysis for VR-NSP11 (A) and LY-NSP11 (B). VR-NSP11 and LY-NSP11 were incubated with R23.1 oligo and each of the seven additional analogs at 15 µM level. DMSO or ethanol: negative vehicle control with NSP11 and R23.1 oligo mixed with DMSO or ethanol; DsRed: negative protein control with DsRed and R23.1 oligo mixed with DMSO. Values represent mean ± SD, n = 3. (C) MTT analysis of the six additional analogs incubated with PAMs for 48 h, with DMSO as the vehicle control. The red dashed line indicates 95% relative viability. The top x-axis illustrates DMSO concentration, expressed as the ratio of DMSO volume to medium volume (vol/vol). The bottom x-axis denotes compound concentrations (µM). Values represent mean ± SD, n = 3. (D) Chemical structures of compounds A8-A2 and Madrasin. (E) Dosage-dependent inhibitory activity of compound A8-A2 (left) and Madrasin (right) to VR/LY-NSP11. Values represent mean ± SD, n = 3.

Fig 3

Binding interaction between NSP11 and its inhibitors. (A) MST analysis of NHS-labeled VR-NSP11 thermal dynamic association with ligands A8 (left) and A8-A2 (right). Values represent mean ± SD, n = 3. (B) NendoU activity analysis with different concentrations of wild-type VR-NSP11 and VR-mNSP11. DsRed protein was used as the negative protein control. Values represent mean ± SD, n = 3. (C) NendoU assay analysis with different concentrations of wild-type LY-NSP11 and LY-mNSP11. DsRed protein was used as the negative protein control. Values represent mean ± SD, n = 3. (D) MST analysis of NHS-labeed VR-NSP11 and VR-mutated NSP11 (VR-mNSP11) protein thermal dynamic association with ligand A8 or A8-A2. Values represent mean ± SD, n = 3. (E) MST analysis of His-tag labeled LY-NSP11 and LY-mNSP11 protein thermal dynamic association with ligand A8 or A8-A2. Values represent mean ± SD, n = 3. (F) Molecular docking analysis illustrating the potential interactions between compound A8-A2 with the catalytic region of wild-type NSP11 and mNSP11. (G) Molecular dynamics simulation highlighting the hydrogen bonds formed between A8-A2 and both wild-type and mNSP11. (H) Interaction fractions of molecular bonds between A8-A2 and both wild-type and mNSP11. (I) Phase diagram from molecular dynamics simulation showing the hydrogen bonds between A8-A2 and both wild-type and mNSP11.

Fig 4

Validation of the NSP11 inhibitor efficacy against PRRSV infection. (A) qRT-PCR analysis of LY, VR, NADC30, SDSU73, and SD16 RNA in PAMs treated with different concentrations of A8-A2. DMSO was used as negative controls. Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. Bars marked with different letters represent statistically significant differences between groups, with each letter indicating a distinct group at a significance level of P < 0.05. (B) Viral titration analysis in TCID₅₀/mL for LY, VR, NADC30, SDSU73, and SD16 in supernatants of PAMs treated as described in (A). Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. (C) Cytotoxicity analysis of different concentrations of Remdesivir incubated with PAMs for 48 h, with DMSO as the vehicle control. The red dashed line indicates 90% relative viability. The top x-axis illustrates DMSO concentration, expressed as the ratio of DMSO volume to medium volume (vol/vol). The bottom x-axis denotes compound concentrations (µM). Values represent mean ± SD, n = 3. (D) qRT-PCR analysis of SD16 RNA in PAMs treated with different concentrations of Remdesivir. DMSO was used as negative controls. Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. (E) Viral titration analysis in TCID₅₀/mL for SD16 in supernatants of PAMs treated as described in (D). Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. (F) Representative immunofluorescence images of PAMs infected with or without PRRSV and treated with DMSO or A8-A2 at 24 h post infection. NC: non-infected cells treated with DMSO. DMSO: infected cells treated with DMSO. A8-A2: infected cells treated with 10–50 μM A8-A2. Blue represents DAPI and green represents PRRSV. Bar  =  100 µM. (G) The percentage of PRRSV-positive cells in images described in (F). Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test.

Fig 5

Conservation of the NendoU catalytic region across PRRSV strains. (A) Neighbor-joining tree representing the whole-genome phylogeny of various PRRSV strains. (B) Sequence alignment of the catalytic regions of NSP11 across different PRRSV strains. Blue marks indicate completely conserved sequences, green marks indicate 80% conservation, and red marks indicate 60% conservation. The key residues critical for NSP11 NendoU catalytic activity are highlighted with red frames. (C) Three-dimensional structural alignment of NSP11 proteins from various PRRSV strains. (D) Pairwise comparison of root mean squared deviation (RMSD) values based on NSP11 protein folding across PRRSV strains. (E) Comparison of changes in the total binding energy during molecular docking and dynamics simulations between NSP11 from various PRRSV strains and A8-A2.

Fig 6

Conservation of the NendoU catalytic region across nidoviruses. (A) Sequence alignment of the catalytic regions of NendoU for different nidoviruses. Blue marks indicate completely conserved sequences, green marks indicate 80% conservation, and red marks indicate 60% conservation. (B) Three-dimensional structural alignment of NendoU-contained proteins for different nidoviuses. (C) Comparison of changes in the total binding energy during molecular docking and dynamics simulations between NendoU-contained proteins from different nidoviruses and A8-A2. (D) Cytotoxicity analysis of CEK cells treated with different concentrations of A8-A2, with DMSO as the vehicle control. The red dashed line indicates 95% relative viability. The top x-axis illustrates DMSO concentration, expressed as the ratio of DMSO volume to medium volume (vol/vol). The bottom x-axis denotes compound concentrations (µM). Values represent mean ± SD, n = 3. (E) qRT-PCR analysis of IBV-MASS RNA contents in CEK cells treated with different concentrations of A8-A2 or vehicle control DMSO. Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. Bars marked with different letters represent statistically significant differences between groups, with each letter indicating a distinct group at a significance level of P < 0.05. (F) Viral titration analysis in TCID₅₀/mL for IBV-MASS in supernatant of CEK cells treated as described in (E). Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test.

Fig 7

Impact of A8-A2 on PRRSV replication and host immune response. (A) PCA of RNA-seq data. D: non-infected cells treated with DMSO. V: PRRSV SD16 infected cells treated with DMSO. A10V, A20V, and A30V: PRRSV SD16-infected cells treated with 10, 20, or 30 µM A8-A2. (B) Biological process analysis of RNA-seq data for the groups described in (A). (C) Differential and GO enrichment analysis of RNA-seq data for the groups described in (A). (D) qRT-PCR analysis of IFN-β expression in PAMs infected with or without PRRSV and treated with DMSO or A8-A2 at 24 h after infection. NC: non-infected cells treated with DMSO. DMSO: infected cells treated with DMSO. A8-A2: infected cells treated with 5–50 μM A8-A2. Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. Bars marked with different letters represent statistically significant differences between groups, with each letter indicating a distinct group at a significance level of P < 0.05. (E) qRT-PCR analysis of viral RNA contents in PAMs infected with or without PRRSV and treated with or without A8-A2 at 0, 8, 12, and 16 h after infection. NC: non-infected cells treated with DMSO. DMSO: infected cells treated with DMSO. A8-A2: infected cells treated with 40 µM A8-A2. Values represent mean ± SD, n = 3. (F) qRT-PCR analysis of IFN-β expression in PAMs infected with or without PRRSV and treated with or without A8-A2 at 0, 8, 12, and 16 h after infection. NC: non-infected cells treated with DMSO. DMSO: infected cells treated with DMSO. A8-A2: infected cells treated with 40 µM A8-A2. Values represent mean ± SD, n = 3. (G) Ratio of viral RNA contents from (E) to IFN-β expression from (F) in PAMs infected with or without PRRSV and treated with or without A8-A2 at 0, 8, 12, and 16 h after infection. NC: non-infected cells treated with DMSO. DMSO: infected cells treated with DMSO. A8-A2: infected cells treated with 40 µM A8-A2. Values represent mean ± SD, n = 3. (H) qRT-PCR analysis of IFN-β in HEK293T cells expressing either DsRed or DsRed-PRRSV-NSP11, transfected with PBS control or Poly(I:C) and treated with DMSO control or 40 µM A8-A2. “+” represents presence and “−” indicates absence of proteins or chemicals. Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test.

Fig 8

The effect of A8-A2 on RNA spliceosome activity. (A) Analysis of alternative splicing events including SE, MXE, A5SS, A3SS, and RI from RNA-seq data. D: non-infected cells treated with DMSO. V: PRRSV SD16-infected cells treated with DMSO. A10V, A20V, and A30V: PRRSV SD16-infected cells treated with 10, 20, or 30 µM A8-A2. (B) Cytotoxicity analysis of Pladienolide B at various concentrations in PAMs at 48 h, with DMSO as the vehicle control. The red dashed line indicates 90% relative cell viability. Values represent mean ± SD, n = 3. (C) qRT-PCR analysis of SD16 RNA in PAMs treated with different concentrations of Pladienolide B, with DMSO as the negative control and A8-A2 as the positive control. Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. Bars marked with different letters represent statistically significant differences between groups, with each letter indicating a distinct group at a significance level of P < 0.05. (D) Viral titration analysis (TCID₅₀/mL) of SD16 in the supernatant of PAMs treated as described in (C). Values represent mean ± SD, n = 3. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test.